KR100528664B1 - Composition for Diagnosing Diabetic Retinopathy and a kit for Diagnosing Diabetic Retinopathy comprising the same - Google Patents

Abstract

The present invention relates to a diagnosis agent for diabetic retinopathy, and more particularly, to an immunoglobulin A (Immunoglobulin A) protein capable of diagnosing the diabetic retina, a diagnostic kit comprising an antibody of the protein and a diagnostic method thereof. . The present invention can be used for diagnosing diabetic retinopathy.

Description

Composition for Diagnosing Diabetic Retinopathy and a kit for Diagnosing Diabetic Retinopathy comprising the same}

The present invention relates to a diagnosis agent for diabetic retinopathy, and more particularly, to an immunoglobulin A (Immunoglobulin A) protein capable of diagnosing the diabetic retina, a diagnostic kit comprising an antibody of the protein and a diagnostic method thereof. .

In general, diabetes is a complex metabolic disease that causes microvascular lesions and causes a wide range of disorders in systemic tissues including the eye and is the most important systemic disease affecting the eye (Lee Tae-hee, Choi Young-gil, Diabetic vascular complications , Seoul: Korea Medicine (1993). Among them, diabetic retinopathy is one of the most serious complications, and it has become an important problem as the life expectancy and length of disease of diabetic patients are prolonged due to improvement of living standard and treatment level (Klein R. et al , Arch Ophthalmol. 102: 520-532 (1984)). Diabetic retinopathy is divided into non-proliferative diabetic retinopathy, in which retinal lesions due to vascular disorders are confined in the retina, and proliferative diabetic retinopathy in which neovascular tissue penetrates into the vitreous cavity from the retina (Green, In: Spencer WH, ed. Ophthalmic Pathology: an atlas and textbook.4th ed.Philadelphia: WB Saunder; 1124-1129 (1996)). Diagnosis of diabetic retinopathy is made by observing characteristic structural changes in the fundus. Visual damage due to diabetic retinopathy is due to vitreous hemorrhage in proliferative diabetic retinopathy and macular degeneration along with tractional retinal detachment of the macula. The usefulness of laser therapy along with surgical treatment is well known (Diabetic Retinopathy Study Report Number 14: Int Ophthalmol Clin. 27: 239-253 (1987)). This treatment can be done at an appropriate stage to prevent vision loss with minimal side effects. Therefore, diabetic retinopathy and diagnostic efforts should be made through frequent tests to determine whether or not the stage of surgical treatment has already been reached. However, until now, only the examination by fundus photography performed in ophthalmology as a diagnostic method is possible. Therefore, it is difficult to diagnose early, and often miss the timing of prevention and surgery. Therefore, the present invention has devised a method for diagnosing diabetic retinopathy easily in the blood. At present, there is no diagnostic method for diabetic retinopathy through blood, and the present inventors used proteomics to find proteins with changes in blood and apply them to diagnosis. This protein has been shown to be a quantitative change in patients with diabetes alone and even diabetic complications to complete the invention using accurate quantitation by immunological methods.

The present invention solves the above-mentioned problems, and the object of the present invention is to provide a diagnostic agent for diabetic retinopathy.

Another object of the present invention to provide a diagnostic kit for diabetic retinopathy comprising the diagnostic agent.

Another object of the present invention is to provide an easy and simple method for diagnosing diabetic retinopathy.

In order to achieve the above object, the present invention provides an iminoglobulin A protein and its protein fragments effective for diagnosing diabetic retinopathy.

The sequence found through the protein analysis of the present invention corresponds to the constant region of the iminoglobulin A heavy chain. The iminoglobulin A protein has a heavy chain and a light chain, and each chain has a variable region so that various sequences are possible. Thus, a protein having a sequence that can turn out to be an immunoglobulin A protein is possible.

Therefore, the iminoglobulin A protein of the present invention can be a variety of sequences in addition to the sequence of the heavy chain of SEQ ID NO: 1 below

The sequence of the H chain of Ig is as described in SEQ ID NO: 1 below.

In addition, in the present invention, the protein fragment of the iminoglobulin A may include several fragments including the peptide of SEQ ID NO: 2.

In addition, the present invention provides an antibody that binds to the above protein, wherein the antibody is preferably polyclonal or monoclonal, but more preferably monoclonal antibody.

The present invention also provides a kit for diagnosing diabetic retinopathy comprising the above antibody.

Kit of the present invention is composed of a kit for diagnosing diabetic retinopathy characterized in that it further comprises a protein labeled with an anti-iminoglobulin A antibody with enzyme peroxidase, alkaline phophatase, biotin, etc. Lose.

The remaining reagents used in the diagnostic kit in the present invention can be easily selected from the components used in the general diagnostic kit.

In another aspect, the present invention comprises the steps of treating the antibody with an anti-iminoglobulin A antibody labeled with a vitreous or blood sample and peroxidase, alkaline phosphatase, biotin and the like; And by measuring the absorbance of the complex provides a method for diagnosing diabetic retinopathy when showing a lower absorbance (ELISA value) than a patient suffering from diabetes alone.

The present invention also provides the nucleotide of SEQ ID NO: 4 encoding the iminoglobulin A gene of SEQ ID NO: 3 and the peptide of SEQ ID NO: 2 encoding the iminoglobulin A protein.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention confirms that the protein changes in the blood from the fact that the iminoglobulin A is increased in the intraocular vitreous of diabetic retinopathy compared to the normal vitreous, and in the blood, the diabetic retinopathy It has been found that noglobulin A has decreased, and this relates to a diagnostic agent for diabetic retinopathy completed by immunological methods.

In order to achieve the above object, the present invention first analyzed the protein group having a special change in diabetic retinopathy by proteomics method. By analyzing protein types and quantitative changes of vitreous changes in normal and diabetic retinopathy patients, the following novel phenomena were identified, and the appropriate protein changes were identified in the blood and applied as an appropriate immunological method for diagnosis of diabetic retinopathy. Diagnosis kits were prepared: First, the intraocular vitreous was collected from diabetic patients and diabetic retinopathy patients with normal persons as a control group to obtain a protein group showing qualitative and quantitative differences through two-dimensional gel separation and image analysis. MS and Q-TOF analyzers were used to identify the identity of the groups. The protein whose changes were observed and confirmed was identified as iminoglobulin A, and an increase in iminoglobulin A, which is rarely observed in normal vitreous, was observed in the vitreous of diabetic retinopathy. There is no report of an increase in iminoglobulin A in diabetic retinopathy. ; Second, this quantitative change showed a quantitative change in the blood. When diabetic blood was used as a control, iminoglobulin A decreased in the blood of diabetic retinopathy. This result has not been reported yet; Third, the abundance level of the protein was adopted by making a kit through an immunological method, which was easy to use, sensitive and high in accuracy.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1 Ocular Vitreous Sample Treatment for Proteomic Analysis

Diabetic retinopathy is one of the complications of long-term diabetes. This disease causes a lot of new blood vessels with incomplete vascular structure, causing bleeding in the intraocular vitreous, causing abnormalities in the retina, depending on the extent of vision loss and loss. In the present invention, through the protein analysis of the normal control group and diabetic retinopathy patient, the disease-causing factor was searched and the information about the protein expressing the disease state was achieved. Protein analysis uses the latest proteomics method. First, the ocular vitreous was easily processed for application to proteomic methods. The vitreous contains an excess of hyaluronic acid, a high molecular sugar. However, the sugar was found to have a significant adverse effect on protein isolation. Therefore, a method for effectively removing it has been devised (see FIG. 1). In this method, 4 ml of vitreous is diluted with 16 ml of distilled water and placed in a tube with 1,000,000 cut-off membrane. Centrifugation is carried out for 2 hours at 8,000 rpm at 4 ° and repeated three times. Filtered using the difference. Unbound protein was concentrated in a tube with 10,000 cut-off membrane by centrifugation at 4,000 rpm at 4 ?? and then used for analysis. The method of removing the high molecular sugars solved the problem of poor separation at low pH, thus enabling a very effective analysis.

Example 2 Investigation of Intraocular Changed Protein Group in Normal and Diabetic Retinopathy

Only protein from each vitreous was extracted and concentrated to an intravitreal protein concentration of 1 mg / mL and used for analysis. The process first separated the protein in two dimensions, a stepwise separation using two different properties. The first step is the electrical stimulation of the protein, the protein shift according to the pH of the protein element (IEF, pH3-10), the second step is the protein on acrylamide gel (8-18%) according to the molecular weight of the protein Was moved. One-dimensional electrophoresis (protein shift according to pH) was moved for 12 hours at a current of 50 mA per gel, and two-dimensional electrophoresis (protein shift according to molecular weight) was electrophorically shifted to 50 mA per gel on polyacrylamide for 6 hours. The transferred protein was stained with Coomaasie Brilliant Blue-250 dye and silver staining method to confirm its presence, and then the difference between normal protein and intravitreal protein of diabetic retinopathy was analyzed by computer image analysis software Phoretix (Nonlinear dynamics, UK ). As a result of analyzing the proteins of the two groups, there was a protein group showing a difference (see FIGS. 2 and 3).

Example 3 Identification of Proteins Existing in Serum and Diabetic Retinopathy in Patients Only Diabetic

 The protein was identified by MALDI-TOF and Q-TOF analyzer to find a protein having a quantitative or qualitative difference (see FIG. 4). This led to a decrease in the amount of iminoglobulin A in diabetic retinopathy blood.

Example 4 Diagnosis of Diabetic Retinopathy by Enzyme Immunoassay

The purpose of this study was to determine whether serum of diabetic retinopathy could be identified by diabetic enzyme immunoassay (ELISA) using anti-iminoglobulin A antibody. Serum from 10 normal patients, 45 diabetic patients without diabetic retinopathy and 86 diabetic retinopathy patients obtained from the hospital was used. First, 100 μl of anti-iminoglobulin A (Koma, Korea) dissolved in a coating buffer (50 mM NaHCO ₃ , pH9.0) per well on an EIA 96-hole plate at a concentration of 10 ug / ml (1 ug of antibody protein per well) ) Was coated for 1 hour at room temperature, washed twice with 400ul of PBST for 10 minutes, and then coated with PBS containing 1% BSA. Add 100ul of serum of the patient diluted with PBST buffer, react for 1 hour, wash 5 times with PBS, and dilute 100ml of anti-imoglobulin A antibody (KOMA Biotech Inc., Korea ) labeled with peroxidase. The reaction was carried out for 1 hour. After the reaction, the mixture was washed three times with PBS, and then 100 μl of 0.1 M citrate-phosphate buffer (pH 4.9) containing 1 mg / ml OPD (o-phenylenediamine dihydrochloride) and 0.03% H ₂ O ₂ was added thereto. After the reaction at room temperature for 20-30 minutes, 100ul of 3M sulfuric acid was added to stop the reaction, and the absorbance was measured at 450 nm using an ELISA reader. This absorbance was determined by applying the conversion and dilution ratio through the standard titration curve to determine the amount of iminoglobulin A per unit volume (ml) of blood (see Fig. 5). The ELISA results showed that the distribution of measured values of iminoglobulin A in serum of normal subjects was 131.2-298.7 mg / dL, and the distribution of measured values of iminoglobulin A in serum of patients with diabetes alone was 226.5-771.9 mg / dL. The distribution of measured values of iminoglobulin A in the sera of retinopathy patients was 105.3-557.2 mg / dL. This is shown in Table 1 as an average value. The measured mean value of iminoglobulin A is 217.6 ± 2.1 mg / dL in normal, 457.5 ± 51.6 mg / dL in patients with diabetes alone, 244.4 ± 17.1 mg / dL in non-proliferative patients with diabetic retinopathy and proliferative. 278.6 ± 23.6 mg / dL. From these results, it can be seen that there is a large amount of iminoglobulin A in serum in patients with diabetes alone, and the difference in serum iminoglobulin A between patients with non-proliferative and proliferative diabetic retinopathy. It can be seen that it does not show. In diabetic patients, if the amount of iminoglobulin A is less than 400 mg / dL of ELISA, diabetic retinopathy is positive, and 72 out of 86 patients are diagnosed as patients. The diagnostic sensitivity was about 83.7%. In addition, the rate of diabetic patients without retinopathy was 22 out of 45 patients, which showed a diagnostic specificity of 48.9% (see Table 2).

TABLE 1 Average value of iminoglobulin A in serum of normal and patient serum by ELISA

Average of IgA Conc. (Mg / dL) Healthy 217.6 ± 82.1 Diabetes (DM) 457.5 ± 151.6 Diabetic Retinopathy (DMR) Non-proliferative (NPDR) 244.4 ± 117.1 Proliferative (PDR) 278.6 ± 123.6

[Table 2] Diagnosis of Diabetic Retinopathy by ELISA

Reference 400 mg / dL (cut off)

Healthy people DM without retinopathy DM with retinopathy 400 mg / dL or more 0 22 14 400 mg / dL or less 10 23 72 Total 10 45 86

The present invention is a technology that can easily diagnose diabetic retinopathy, a complication of diabetes. To date, there are no commercially available efficient diagnostic agents for diabetic retinopathy. Diagnosis of diabetic retinopathy is wholly visited to the hospital by an ophthalmologist examination. Therefore, early diagnosis is not possible when diabetics do not feel abnormal vision due to subjective symptoms and do not undergo regular eye examination. Since the development of this diagnostic agent is characterized by a simple blood test, it is very useful in that the complications can be checked before the ophthalmologic examination. In particular, the present invention has the advantage of being easy and inexpensive and easy to handle for a large number of diabetic patients who visit the medical examination or internal medicine by adopting the ELISA method using 96 holes that can be examined in large quantities. Immunological methods are used to provide excellent accuracy and precision. In conclusion, this invention is useful for early diagnosis and screening in diabetic retinopathy, and can help to determine the timing of treatment and delay the disease to severe diabetic retinopathy.

Figure 1 shows a schematic view of the eye glass pretreatment for application to proteomics.

Figure 2 shows the gel photograph of the CBB staining the ocular vitreous protein after two-dimensional electrophoresis. The area indicated is not present in the normal vitreous eye but shows an increased amount in the vitreous of diabetic retinopathy eye.

Figure 3 shows a CBB stained gel picture after the two-dimensional electrophoresis of the serum protein of the patient with diabetes and diabetic retinopathy patients. Marked areas are present in patients with diabetes alone, but serum in patients with diabetic retinopathy shows reduced amounts of protein.

FIG. 4 shows the mass spectrum (A) of trypsinized peptides in the proteins of the indicated region of FIG. 2 and the amino acid sequence of one of the peptides (B) using MALDI-TOF and Q-TOF analyzers.

Figure 5 is a standard titration graph of the iminoglobulin A standard solution 0, 15.6, 31.25, 62.5, 125, 250, 500 ng / ml concentration and the absorbance measurements after ELISA reaction. This graph can be used to calculate the unknown amount of iminoglobulin A.

<110> EYEGENE CO., LTD. <120> Protein for Diagnosing Diabetic Retinopathy <150> KR102002041771 <151> 2002-07-16 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 353 <212> PRT <213> Homo sapiens <400> 1 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys Ser Thr 1 5 10 15 Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110 Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115 120 125 Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn 130 135 140 Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe 145 150 155 160 Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu 165 170 175 Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys 180 185 190 Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr 195 200 205 Pro Glu Ser Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn 210 215 220 Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu 225 230 235 240 Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser 245 250 255 Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro 260 265 270 Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly 275 280 285 Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp 290 295 300 Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu 305 310 315 320 Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro 325 330 335 Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys 340 345 350 Tyr <210> 2 <211> 10 <212> PRT <213> Homo sapiens <400> 2 Trp Leu Gln Gly Ser Gln Glu Leu Pro Arg 1 5 10 <210> 3 <211> 1059 <212> DNA <213> Homo sapiens <400> 3 gcaagcttga ccagccccaa ggtcttcccg ctgagcctct gcagcaccca gccagatggg 60 aacgtggtca tcgcctgcct ggtccagggc ttcttccccc aggagccact cagtgtgacc 120 tggagcgaaa gcggacaggg cgtgaccgcc agaaacttcc cacccagcca ggatgcctcc 180 ggggacctgt acaccacgag cagccagctg accctgccgg ccacacagtg cctagccggc 240 aagtccgtga catgccacgt gaagcactac acgaatccca gccaggatgt gactgtgccc 300 tgcccagttc cctcaactcc acctacccca tctccctcaa ctccacctac cccatctccc 360 tcatgctgcc acccccgact gtcactgcac cgaccggccc tcgaggacct gctcttaggt 420 tcagaagcga acctcacgtg cacactgacc ggcctgagag atgcctcagg tgtcaccttc 480 acctggacgc cctcaagtgg gaagagcgct gttcaaggac cacctgaccg tgacctctgt 540 ggctgctaca gcgtgtccag tgtcctgtcg ggctgtgccg agccatggaa ccatgggaag 600 accttcactt gcactgctgc ctaccccgag tccaagaccc cgctaaccgc caccctctca 660 aaatccggaa acacattccg gcccgaggtc cacctgctgc cgccgccgtc ggaggagctg 720 gccctgaacg agctggtgac gctgacgtgc ctggcacgtg gcttcagccc caaggatgtg 780 ctggttcgct ggctgcaggg gtcacaggag ctgccccgcg agaagtacct gacttgggca 840 tcccggcagg agcccagcca gggcaccacc accttcgctg tgaccagcat actgcgcgtg 900 gcagccgagg actggaagaa gggggacacc ttctcctgca tggtgggcca cgaggccctg 960 ccgctggcct tcacacagaa gaccatcgac cgcttggcgg gtaaacccac ccatgtcaat 1020 gtgtctgttg tcatggcgga ggtggacggc acctgctac 1059 <210> 4 <211> 30 <212> DNA <213> Homo sapiens <400> 4 tggctgcagg ggtcacagga gctgccccgc 30

Claims (7)

Having a lesion of diabetic retinopathy in diabetic patients by measuring the concentration of iminoglobulin A protein or fragment thereof in blood of a diabetic patient, comprising an antibody that binds to the iminoglobulin A protein or a fragment thereof described in SEQ ID NO: 1 Diabetic retinopathy diagnostic composition for diagnosing a patient. The composition of claim 1, wherein the protein fragment comprises a peptide sequence set forth in SEQ ID NO: 2. delete Kit for diagnosing diabetic retinopathy comprising the composition of claim 1. The method of claim 4, wherein the kit further comprises an anti-iminoglobulin A antibody labeled with a label selected from the group consisting of enzyme peroxidase, alkaline phosphatase, and biotin. Retinopathy diagnostic kit. delete delete